Cell detachment with trypsin/EDTA: the biochemistry going on when you take cells off!

[@thebumblingbiochemist]

now a fun note on anti-trypsin, which has a fun name - it’s a SERPIN. These are not snakes, they’re “serine scissor safety guards.” Their name comes from SErine PRotease INhibitors and they act as “fake substrates” that get stuck in the protease when it tries to cut them - it’s able to cut them but it can’t release them because, once cut, the serpin can (still bound to trypsin) shape-change, drag trypsin along with it, and move the trypsin’s active site out of whack in the process. An example of a serpin you might have heard mentioned in commercials for emphysema treatments is alpha-1-antitrypsin - in the bloodstream, it protects tissues from the serine protease elastase, which a type of immune cells called neutrophils secrete into sites of inflammation to break down connective tissue so that blood cells have an easier time getting in to go to work repairing damage. alpha 1-antitrypsin acts as a sort of “moat” that limits elastase’s area of action so it doesn’t just go chewing up connective tissue throughout your body. But smoking can modify this serpin so that it fails to do its job adequately, so the moat is breached and elastase is able to chew up the lungs. Some people have a genetic alpha-1-antitrypsin deficiency, so they’re more at risk for emphysema even in they don’t smoke. alpha 1-antitrypsin is just one example of more than 30 serpins have been discovered in humans. A couple others are antithrombin, which keeps thrombin in check during clot formation and antiplasmin, which inhibits plasmin so that clots can be disassembled. Here’s that cool page about cadherins: Cells Get Stick with Calcium, UIUC Theoretical and Computational Biophysics Group: www.ks.uiuc.edu/Research/cadherin/ Some helpful resources: Subculturing Adherent Cells, ThermoFisher Scientific/Gibco: www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/subculturing-adherent-cells.html Useful numbers for cell culture, ThermoFisher Scientific/Gibco: www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html Cell Dissociation Protocol using Trypsin, Millipore Sigma: www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/cell-dissociation-with-trypsin Evolution of cell culture surfaces, John A. Ryan, Ph.D., Corning Life Sciences: www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/evolution-of-cell more on HEK293 cells: blog form: bit.ly/hekcells ; KZbin: kzbin.info/www/bejne/qn3XfICsj5eta7c   more about culture media: blog: bit.ly/cell_culture_media ; KZbin: kzbin.info/www/bejne/bniuf2lmf56geZY   more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@dennyhamrick2552]

Hey, I just wanted to say: I started my first tech job out of undergrad recently and your videos (and website content) have been unbelievably helpful, especially in understanding how each reagent in a process actually functions. Thanks so much for all the effort you put in; it is very appreciated

[@thebumblingbiochemist]

Soooooo happy to hear! Thank you so much. I really appreciate you saying that and wish you the best of luck in your new job!

[@jennifer5nogueira]

Hi!! I'm a junior research in Brazil and I really love your work! One doubt: when culturing adherent cells, the tripsinization should only make them round and I finish the job by gently hitting the flask or let the tyrosine totally detach them from the bottom of the flask? I'm always afraid of letting them for a long period with trypsin, so I usually end up hitting the flask. Thanks!

[@thebumblingbiochemist]

I normally would do just like you

[@alfredogonzalez8735]

When trypsinizing, once we add media and deactivate the trypsin, do we need to centrifuge and remove the media and suspend in fresh media? In my lab I've just been adding media, aspirating, and then adding half the volume to two different flasks that already have some fresh media. Just not sure if the centrifugation is necessary.

[@thebumblingbiochemist]

I always removed the trypsin-containing media so it didn't affect anything later on.